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t




Ligation to concatemers

t




Packaging in phage lambda heads




I
Infection of E. and selection of a set of
cosmids for overlap recombination

t
-=-+iiF˜
I
Cotransfection of insert fragments

1™ ™ ™

,
cl
In vivo reconstitution of virus by overlap recombination


I” ” I

47
de Wind, van ZI$ and Berm
48
ments resulted m efficient generation of virus progeny. This confirms that the
process of homologous recombinatton 1svery efficient (see Note 3). Virus thus
regenerated is mdistmguishable from wild-type PRV, both on the basis of
restriction enzyme analysis as well as by phenotypic analysis (virulence and
pathogemcity for pigs [2/). As described next, these mdividual vu-us subclones
are easily amenable to large-scale systematic mutagenesis.
1.2. The Generation of Oligonucleo tide Insertion Mu tan ts
of the Virus Using Large Cloned Subgenomic Regions
Each of the cloned subgenomic regions of the vuus can now be subjected to
mutagenesis m vitro to effictently generate large numbers of mutant deriva-
tives of the cosmid clone. This is subsequently followed by overlap recombi-
nation, after cotransfection of each mutant cosmid derivative together with the
overlappmg cloned virus genomic fragments, leading to the regeneration of
virus carrying the specific mutation (Fig. 1B).
To systematically mactivate genes located on these large plasmids, we have
modrfled common ohgonucleotide insertion mutagenesistechmques(refs. 5 and 6;
Fig. 3). These modifications enable insertion, at a random site within such a
large plasmid, a palmdromic double-stranded ohgonucleotide that contains stop
codons in all SIX reading frames as well as a restriction site that normally is not
present in that cosmid (and preferably also not in the rest of the vnus genome).
Presence of this oligonucleotide m any orientation within a coding region will,
as a consequence of the insertion of a stop codon m the reading frame, lead to
termination of translation of the messenger RNA, thus elimmatmg expression
of that gene (see Note 4). As an example, for the mutagenesis of PRV clones,
we used the 20-mer ollgonucleotide S-TAGGCTAGAATTCTAGCCTA-3™,
which contains an EcoRI site (underlmed, this site is absent from the PRV
genome) and amber stop codons in all reading frames (italicized, see Note 5).
The pivotal step m the procedure of mutagenesis is the lmeartzation of the
clone at a random site, leaving a blunt-ended linear molecule that IS suitable
for the insertion of the double-stranded ollgonucleotide This lmearizatron 1s
achieved by very partial digestion using restriction enzymes that have a 4-bp
recognition site and that leave a blunt end after cleavage (see Note 6) The
presence of Ethidmm bromide m the dtgestion mixture, which mhtbtts mul-
tiple cuts of the same DNA molecule (7), will result in an increased proportion

Fig 3. (opposzte) Outline of the procedurefor the insertion of the ohgonucleotide at
unique and random srtes within a cosmld clone. This IS followed by overlap recombma-
tlons, each mcludmg a mutant cosmld derivative and the three overlappmg cloned frag-
ments. A viable virus mutant IS generated that lacks the expression of a smgle gene, If
the gene mto which a specific ohgonucleottde is inserted, IS not essential for the virus
Linearization of one of the cosmid clones at quasi-random sites by
fine-cutting restriction enzymes in the presence of Ethidium bromide
t




l
Insertion of an oligonucleotide, containing stopcodons in all reading frames,
and a unique restriction site (t), followed by transformation of E.




I
Selection of a set of mutant cosmid derivatives for overlap recombination




Overlap recombinations io generate virus mutants
de Wind, van Zijl, and Berns
50




- LINEAR (45 kbp)




Fig. 4. A 4%kbp cosmid clone that is linearized at random sites by cleavage with
blunt cleaving restriction sites with four-bp specificity (FnuDII: CGCG; HaeIII:
GGCC; RsaI: GTAC), in the presenceof Ethidium bromide. OC, open circular DNA,
CC, covalently closed DNA. Also indicated: (full length) linear DNA and multiply
digested (degraded) linear DNA fragments.

(up to about 50%) of linearized plasmids. An agarose gel of such digests is
shown in Fig. 4. After purification of the full-length linearized plasmids, the
double-stranded oligonucleotide is inserted at the site of linearization. After
recirculation of the resulting recombinant plasmids, E. coli is transformed
and a library is obtained of recombinant plasmids, each carrying the inserted
oligonucleotide at a unique and random site, essentially as depicted in Fig. 3.
Mutant plasmids thus obtained are characterized for integrity and site of
insertion of the oligonucleotide by restriction enzyme analysis (see also Note
7). A set of mutant virus strains is subsequently reconstituted by overlap
recombination using a selected set of mutant plasmid inserts, together with
overlapping cloned virus fragments, as described in Section 1.1.1. (see Fig. 3
see also Note 8). Viable virus mutants thus obtained completely lack the
expression of a single gene (as exemplified in ref. 5) and are amenable to
phenotypic study in tissue culture (see refs. 8-14). In addition, they can also
be used to study the effect of the gene on virulence, pathogenicity, and
immunogenicity of the virus in animals (6,13-Zli). Furthermore, oligonucle-
otide-bearing cosmid clones can be used as the starting point for further modi-
fication of the genome, e.g., for the generation of specified deletions into the
virus genome or for the insertion of heterologous genes. We will give an
outline of the different steps involved.
Charactenzatlon of a Herpeswrus Genome 51

1.3. The Generation of Gene-Specific Probes Flanking
the Inserted Oligonucleotide in Each Mutant
to Explore the Virus Genome
The set of virus clones, each carrying an insertion of the oligonucleotide at a
different, random site also permits easy access to gene-specific cloned short
DNA fragments. These are subcloned after digestion of the ohgonucleotide-
bearing large virus clones with the restriction enzyme for which a unique site IS
present in the Inserted ohgonucleotide plus a second restriction enzyme havmg
a 4-bp specificity that most likely cleaves nearby.
These subcloned short virus fragments are useful for a number of analyses
(see Fig. 1C and ref. 6):
1. As probes in cross hybridization experiments with DNA of a prototype herpesvi-
rus to gain information on the genomic relationship between both viruses,
2. As probes in Northern blotting experiments on RNA isolated from infected cells
to acquire information on location, size, and class of transcripts encoded by that
specific region of the virus genome; and
3. For direct sequence analysis to identify the gene inactivated in each specific oli-
gonucleotide insertion mutant
Given the scope of this chapter, we will only provide here an outline of the
procedures involved m these experiments
The methodology described m this chapter requires careful planning and the
labor of preparation and characterization of a cosmid library of the virus
genome and of the subsequent mutagenesis procedure. The only prerequisites
to the methodology are that viable virus be obtained by transfection of the
virus genome (this may be assayed using the protocol given m Section 3 1.16.).
In addition, a precise restriction map of the virus for one, or preferably more,
diagnostic enzymes must be established in advance. In conclusion, if the herp-
esvirus under study is less well known, such a set of virus clones for overlap
recombination, followed by oligonucleotide insertion mutagenesis, will be a
valuable tool to rapidly perform a thorough analysis of gene structure and gene
function of the virus.

2. Materials
2.1. Cloning the Virus Genome as a Cosmid Library
1 10X DNase buffer 500 mMTris-HCI, pH 7.4, 10 mn/f MnC12, 1 mg/mL bovine
serum albumin. A stock solution of 2.50 ug DNaseI/mL is made by dissolving
lyophilized enzyme in 1X DNase buffer. The stock is divided in small ahquots
and frozen at -20°C Use each tube only once
2. Phenol: Add 8-hydroxyquinohne to 0.1% of phenol, which is liquefied by warm-
ing. Extract repeatedly with equal volume of 0 lMTris-HCl, pH 8 0, until the pH
de Wind, van ZIJ, and Berns
52
of the aqueous phase is greater than 7 6. Aliquot in 50-mL conical tubes, and
store m the dark at -20°C m 5 mL buffer.
Phenol chloroform isoamyl alcohol MIX 25 vol phenol with 24 vol chloroform
3
and 1 vol isoamyl alcohol
3M Sodium acetate pH 5.2 and pH 7 0
4
TE. 10 mM Tris-HCI, pH 7.4, 1 mM EDTA pH 8.0 Sterilize both solutions by
5.
autoclavmg.
Absolute ethanol
6
Isopropanol
7.
70% v/v Ethanol
8
Running buffer (TAE) Usually made as a 50X concentrate™ 242 g Tris base, 57 1
9
mL glacial acetic acid, 100 mL 0 5M EDTA pH 8.0/L. Prior to use, this concen-
trate is diluted 50X and 50 ˜.ILof a IO mg/mL ethidmm bromtde solutton IS added
per liter. Warning: Ethidmm bromide IS mutagenic. Wear gloves while handling
solutions Also, store ethidmm bromide solution shielded from light Use a good,
preparative, quality agarose
10 5X sample buffer. 50% (w/v) sucrose, 1% (w/v) SDS, 0 1% Bromophenol blue
11 Stze markers phage lambda DNA and phage lambda DNA, digested with HlndlII,
can best be purchased
12. Glycerol gradients. 50 mM Tris-HCl, pH 8 0, 300 mM NaCl, 1 mA4 EDTA pH
8 0, 15% v/v glycerol, and 50 mMTrts-HCl, pH 8 0,300 mMNaC1, 1 mMEDTA
pH 8 0,40% v/v glycerol
13. Acid trrturatlon buffer for preparing transformation-competent E coli* 100
mMCaCl,, 70 mM MgCll, 40 mM sodmm acetate pH 5 5 Filter sterilize The
solution should be used fresh and be ice cold. Note: Preferably use disposable
plasticware prermse glassware thoroughly. Use the purest available water for
all solutions. Any recA-(recombmation defictent) strain of E co11 is suitable
14 Media for E colz. SOB 20 g Bacto tryptone, 5 g yeast extract. 0 58 g NaCl, 0 2 g
KCl/L Autoclave, cool to 60°C and add 20 mL of a 1M sterile MgS04 solution/L
SOC. SOB to which 20 mL/L of a 1M sterile glucose solution IS added after autoclav-
mg. LB* 5 g NaCI, 5 g yeast extract, 10 g tryptone, 0 3 mL 1OM NaOH/L Autoclave.
For LB agar: Add 15 g Bacto agar/L prior to autoclavmg For ampiclllm contaimng
medmm or plates, Add 0 00 1 vol of a filter sterilized aqueous solution of 100 mg/mL
Na ampicillin in HZ0 Medmm must be cooler than 55°C for the addition of amplclllm
15 Plasmtd isolation. solutton I 25 mM Tris-HCl, pH 8 0, 10 mM glucose, 10 mM
EDTA, pH 8 0, 0 02 w/v% NaNs Warning: NaN3 IS extremely toxic. Can also
be prepared as 10X stock. Just before use, add lysozyme to a final concentration
of 5 mg/mL. Solution II 0 2M NaOH (freshly diluted from a 1OM stock), 1%
SDS. Solutron III 3M potassium acetate and 115 mL glacial acetic acid/L Store
solutions at 4°C. In addition, isopropanol, phenol chloroform isoamyl alcohol,
10 mg/mL RNaseA solution m water, boiled for 5 mm to Inactivate DNase con-
tamination This solution IS ahquoted and frozen at -20°C
16 Transfectlon: HBS buffer 140 mMNaCI,5 mMKC1, 0 75 mMNa*HPO,, 6 mM
dextrose, 25 mMHEPES m double distilled (or milhQ) water Shake the solution
53
Characterization of a Herpesvirus Genome
well to saturate rt with CO, (from air) and adJust the final pH to precisely pH 7 05
at room temperature usmg 0 5N NaOH Sterilize using a 0 22-u filter, aliquot,
and store at -20°C Other solutions. 2 5M CaCl,, sterilized by filtration, aliquoted
m I-mL quanttties, and stored at -20°C and 0.1X TE 1 mMTrts-HCl (pH 8.0),
0.1 mMEDTA (pH 8.0), filter sterilized, ahquoted, and stored at 4°C.
17. Cell culture a cell lme permissive for vnus growth (preferably well trans-
fectable), growth medium, growth medmm + 15% (w/v) glycerol, growth medium
+ 1% (w/v) methylcellulose as solidtfymg agent. Other cell culture requirements
are PBS, trypsm, dtsposables, and so on.
18. DNA tsolatton from infected cells-lysts buffer O.lM Trts-HCI, pH 8 0, 0.2M
NaCl, 5 mMEDTA pH 8.0.0.2 w/v% SDS. Add (just before use) proteinase K to
100 pg/rnL. Protemase K IS kept as a frozen stock m water at 20 mg/mL at -20°C.
19 Other requirements-enzymes* restrtctton enzymes, Calf Intestmal Phosphatase
(CIP), polynucleottde kmase, T4 DNA polymerase, T4 DNA hgase. Nowadays,
enzymes generally come with the appropriate reaction buffers. A neutralized
solution containing each of the four dNTPs at a 2-mM concentration. A cosmid
vector may be obtained from, e.g., Boehringer or Stratagene. A phage lambda
packaging kit A Geneclean (Bio 101) or similar ktt for elutton of DNA frag-
ments from agarose gels. 1-mL syrmges, 25-gage needles. Quick Seal 0.5 x 2-m
ultracentrtfuge tubes Electroelution equtpment. A gradient former.

2.2. Oiigonucieotide insertion Mutagenesis
of individual Cosmid C/ones
1 10X Restrictron enzyme buffers for digestion m the presence of ethidmm bro-
mide. FnuDII: 200 mM Trts-HCI pH 7.5, 80 rnA4 MgCl,, 500 pg/mL ethrdmm
bromtde, HaeIII: 200 mMTrrs-HCl pH 7.5,500 mMNaCl,80 mMMgC12, 50 ug/mL
ethtdmm bromide, RsaI. 200 mMTris-HCl pH 7.5,500 mA4NaCl,80 mA4MgC12,
5 pg/mL ethidium bromide Store aliquoted at -20°C.
2. Other requirements. Commercially available ultracompetent cells Any recA-
(recombination deficient) strain of E coli is suitable. Ultrapure preparative aga-
rose (e g , Agarose NA. Pharmacia). A 10% (w/v) SDS solution.

3. Methods
3.1. Cioning the Virus Genome as a Cosmid Library
The different steps of this procedure are depicted schemattcally in Fig 2.
Virus can be grown, and virus DNA may be isolated. To obtain random

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. 8
( 61 .)



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